The broad, long-term objectives of this proposal are to evaluate the global potential of amino acid transporter ATB/ASCT2 as a selective therapeutic target in human hepatocellular carcinoma {HCC) and to elucidate the signaling mechanisms linking it to liver cancer cell growth and survival. Currently, there are no effective therapies for this cancer and its incidence continues to rise in the United States. ASCT2 is expressed in all human liver cancers examined to date, but is not expressed in normal human hepatocytes. Suppression of ASCT2 expression by inducible antisense RNA in the SK-Hep HCC cell line leads to cell death by the intrinsic apoptotic pathway via a mechanism that is attenuated by osmotic cell swelling. Therefore, the working hypothesis is that ASCT2, via its role in substrate delivery and cellular hydration (swelling) is linked to mammalian target of rapamycin (mTOR)- and phosphatidylinositol-3 kinase (PI3K)- dependent survival signaling pathways, respectively, that impinge upon proteins responsible for apoptotic suppression in all human liver cancer ceils. The aims of this proposal thus addresses the universality and mechanisms linking ASCT2 to hepatoma survival: Specific Aim 1: To evaluate the impact of ASCT2 post-transcriptional gene silencing (PTGS) on the growth and viability of six human hepatoma cell lines, representing a broad spectrum of differentiated phenotypes. Research Plan: Several individual candidate small interfering RNA (siRNA) specific to ASCT2 will be transfected into all six human hepatoma cell lines, and assessed for effects on growth, viability, ASCT2 expression and caspase activation, compared to appropriate control siRNA. Subsequently, adenoviral DMA vectors encoding for control and effective siRNA's (ideally 3) will be constructed and employed in signaling experiments. Off-target siRNA effects in ASCT2 silenced cells will be assessed by examination of a select panel of mRNA, microarray analysis and by the ability of reintroduced ASCT2 to "rescue" them. Alternative/corroborative approaches include use of PTGS methods such as morpholino antisense oligonucleotides or an inducible antisense RNA system. Specific Aim 2: To elucidate the signaling pathways linking ASCT2 silencing to hepatoma apoptosis. Research Plan: Signaling via PI3K-linked (PKB/Akt) and amino acid-dependent (mTOR) pathways as they relate to the pro-apoptotic Bcl-2 family member Bad, or other "mitochondrial gatekeepers", will be investigated pre- and post-ASCT2 silencing through the use of selective biochemical inhibitors and western blot analyses. Putative links established through this approach will be later confirmed by the expression of dominant negative or constitutively active variants of Akt or other signaling proteins. The impact of ASCT2 silencing on cell volume will be assessed by flow cytometry analysis, and reciprocally, the ability of artificial cell swelling and shrinkage to modulate these signaling pathways will also be investigated to better understand the mechanistic link between amino acid transporter function and cancer cell survival.